Erythrocyte adhesion proteins Lu and LW (now termed ICAM-4) are well-defined blood groups, but little is known regarding their membrane function. During erythropoiesis, erythroblasts differentiate within erythroblastic islands surrounding a macrophage. We hypothesize that ICAM-4 mediates interactions between erythroblasts via ICAM-4/alpha4beta1 binding and regulates adhesion of erythroblasts to macrophages via ICAM-4/alphaV binding. Peptides corresponding to areas of ICAM-4 that interact with alphaV and beta1 inhibit erythroblastic island formation. Additionally, we identified a secreted isoform of ICAM-4, which may modulate binding. We and others have shown that ICAM-4 also binds integrins present on endothelial cells, neutrophils and platelets. Hence, we will explore the contribution of ICAM-4 to vascular pathology of sickle cell disease. Lu binds laminins containing the alpha5 chain (laminins 10/11) with high affinity. Importantly, cultured erythroblasts increasingly bind laminin 10/11 from day 6 onwards and the level of binding paralleled increasing expression of Lu. We hypothesize that Lu-laminin adhesion functions during enucleation and/or marrow egress, since alpha5 laminin localizes to subendothelial basement membranes of bone marrow sinusoids. To test our hypotheses we propose to: 1) Examine ICAM-4 function by identifying regions of ICAM-4 involved in alpha4beta1 binding employing site directed mutagenesis and in vitro binding assays; characterize the effect of blocking reagents on formation and dissociation of erythroblastic islands; assess interactions between cells within islands in the presence and absence of blocking reagents using micropipette techniques; measure single adhesion bond strength by dynamic force spectroscopy; and study erythroblastic islands in ICAM-4 knockout mice. 2) Determine function of the Lu-laminin receptor complex by identifying the laminin binding region on Lu; developing blocking antibodies and peptides and testing their effects on nuclear extrusion and reticulocyte generation in vitro laminin 10/11; and by analyzing apoptosis, enucleation, and reticulocytosis in Lu knockout mice. 3) Explore contributions of ICAM-4 to vascular pathology in sickle cell disease by studying effects on vascular blood flow of infusing transgenic/knockout sickle mice with peptides and antibodies directed against ICAM-4 which block adhesion of sickle red cells to endothelial cells. Successful accomplishment of these aims will further our goals of developing a mechanistic understanding of normal erythropoiesis and the pathophysiology of sickle cell disease which could lead to novel therapeutic modalities .